Ketol-acid reductoisomerase enzymes are ubiquitous in nature and are involved in the production of valine and isoleucine, pathways that may affect the biological synthesis of isobutanol. Isobutanol is specifically produced from catabolism of L-valine as a by-product of yeast fermentation. It is a component of “fusel oil” that forms as a result of incomplete metabolism of amino acids by yeasts. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the Ehrlich pathway (Dickinson, et al., J. Biol. Chem. 273, 25752-25756, 1998).
Addition of exogenous L-valine to the fermentation increases the yield of isobutanol, as described by Dickinson et al., supra, wherein it is reported that a yield of isobutanol of 3 g/L is obtained by providing L-valine at a concentration of 20 g/L in the fermentation. In addition, production of n-propanol, isobutanol and isoamylalcohol has been shown by calcium alginate immobilized cells of Zymomonas mobilis (Oaxaca, et al., Acta Biotechnol., 11, 523-532, 1991).
An increase in the yield of C3-C5 alcohols from carbohydrates was shown when amino acids leucine, isoleucine, and/or valine were added to the growth medium as the nitrogen source (WO 2005040392).
While methods described above indicate the potential of isobutanol production via biological means these methods are cost prohibitive for industrial scale isobutanol production. The biosynthesis of isobutanol directly from sugars would be economically viable and would represent an advance in the art. However, to date the only ketol-acid reductoisomerase (KARI) enzymes known are those that bind NADPH in its native form, reducing the energy efficiency of the pathway. A KARI that would bind NADH would be beneficial and enhance the productivity of the isobutanol biosynthetic pathway by capitalizing on the NADH produced by the existing glycolytic and other metabolic pathways in most commonly used microbial cells. The discovery of a KARI enzyme that can use NADH as a cofactor as opposed to NADPH would be an advance in the art.
The evolution of enzymes having specificity for the NADH cofactor as opposed to NADPH is known for some enzymes and is commonly referred to as “cofactor switching”. See for example Eppink, et al. J. Mol. Biol., (1999), 292, 87-96, describing the switching of the cofactor specificity of strictly NADPH-dependent p-Hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens by site-directed mutagenesis; and Nakanishi, et al., J. Biol. Chem., (1997), 272, 2218-2222, describing the use of site-directed mutagenesis on a mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D) resulting in an enzyme having a 200-fold increase in the Km values for NADP(H) and a corresponding decrease of more than 7-fold in those for NAD(H). Co-factor switching has been applied to a variety of enzymes including monooxygenases, (Kamerbeek, et al., Eur. J, Biochem., (2004), 271, 2107-2116); dehydrogenases; Nishiyama, et al., J. Biol. Chem., (1993), 268, 4656-4660; Ferredoxin-NADP reductase, Martinez-Julvez, et al., Biophys. Chem., (2005), 115, 219-224); and oxidoreductases (US2004/0248250).
Rane et al., (Arch. Biochem. Biophys., (1997), 338, 83-89) discuss cofactor switching of a ketol acid reductoisomerase isolated from E. coli by targeting four residues in the enzyme for mutagenesis, (R68, K69, K75, and R76,); however the effectiveness of this method is in doubt.
Although the above cited methods suggest that it is generally possible to switch the cofactor specificity between NADH and NADPH, the methods are enzyme specific and the outcomes unpredictable. The development of a ketol-acid reductoisomerase having a high specificity for NADH as opposed to NADPH would greatly enhance its effectiveness in the isobutanol biosynthetic pathway, however, no such KARI enzyme has been reported.
Applicants have solved the stated problem by identifying a number of mutant ketol-acid reductoisomerase enzymes that have a preference for binding NADH as opposed to NADPH.